Memapsin 2 (beta-secretase, BACE) is the protease that initiated the cleavage of beta-amyloid precursor protein (APR) leading to the production of amyloid-beta (Abeta). It is the major target for the development of inhibitor drugs against Alzheimer's disease (AD). The understanding on the activity, structure and function of memapsin 2 is fundamental for inhibitor design and testing. During the last 5 years of this project, we have established assays, completed kinetic specificity analysis, determined crystal structures and designed generations of inhibitors that have attained high potency, relatively small size, and good selectivity vs. chosen human aspartic proteases, cell permeability and inhibition of Abeta production in transgenic AD mice. For the next period of this project, we propose to further probe memapsin 2 for structure-function information relevant to inhibitor development, to acquire better drug-like properties, to explore new chemistry for inhibitor synthesis and to develop non-transition state inhibitors against newly discovered subsites of memapsin 2. The Aims are: Aim 1. To carry out in-depth memapsin 2 structure-function studies for further structure-based inhibitor design. We propose to investigate the binding affinity of new ligands toward memapsin 2 subsites S7, S6 and S5, the role of active-site cleft 'bottleneck'residues on the inhibitor subsite specificity, cellular inhibition by memapsin 2 inhibitors and the therapeutic limits of memapsin 2 inhibitor drugs. Aim 2. Design and testing transition-state memapsin 2 inhibitors with better in vivo potency and selectivity. We propose to optimize ligand-binding site interactions of lead peptidomimetic inhibitors, design and synthesize novel nonpeptidyl high-affinity ligands, templates and scaffolds, incorporate basic amines and lipophilic functionalities for effective absorption and BBB transport, and improve potency of small molecule nonpeptidyl inhibitors. Aim 3. To develop and test novel memapsin 2 inhibitors targeting to new sites in the protease. We propose to design and test a new class of non-transition state inhibitors targeted at 3 unique subsites, P7, P6 and P5. We will use structure-based design cycle to develop small, potent and selective new inhibitors.